Interleukin-9 and Interleukin-4 Chimeric Antagonist Muteins and Methods of Using Same

ABSTRACT

Chimeric polypeptide antagonists that include an interleukin-4 (IL-4) mutein linked to an interleukin-9 (IL-9) mutein are provided, as are polynucleotides encoding the IL-4 and IL-9 chimeric mutein antagonists. Also provided are methods of using the chimeric mutein antagonists and encoding polypeptides to reduce or inhibit the responsiveness of a cell to a cytokine such as IL-4, IL-9 and/or interleukin-13. Methods using the compositions to treat disorders such as pulmonary disorders (e.g., asthma) also are provided.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application is a divisional application of U.S. application Ser.No. 11/026,396 filed Dec. 29, 2004, now issued as U.S. Pat. No.7,635,754; which claims the benefit under 35 USC §119(e) to U.S.Application Ser. No. 60/612,275 filed Sep. 22, 2004, now abandoned. Thedisclosure of each of the prior applications is considered part of andis incorporated by reference in the disclosure of this application.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to compositions useful for treatingmodulating an immune response, and more specifically to chimericinterleukin-4 (IL-4) and interleukin-9 (IL-9) muteins that act asantagonists of interleukin-4, interleukin-9, and interleukin-13activity, and to methods of using such IL-4 and IL-9 chimeric antagonistmuteins to modulate a cytokine-mediated response of a cell and toameliorate a disorder associated with cytokine-mediated responsiveness.

2. Background Information

Interleukin-9 (IL-9), interleukin-4 (IL-4) and interleukin-13 (IL-13)are cytokines produced by activated T-cells upon antigen stimulation.Asthma is characterized by reversible airflow obstruction and airwayhyper-responsiveness (AHR), associated with an infiltration of thebronchial mucosa with activated T-lymphocytes (T-cells), andeosinophils. These cells, along with resident airway mast cells, secretea variety of cytokines and mediators that play a fundamental role in thepathogenesis of the disease (1,2).

A number of studies suggest that IL-9, as a mediator of Th2-dependentimmune responses, has a role in asthma. Human linkage analysis showed anassociation between the IL-9 gene and elevated serum levels of IgEproduction and airway hyper-responsiveness (3,4). IL-9 transgenic miceexhibit many characteristics of human asthma, and have a strikinglyrobust peribronchial and perivascular eosinophilia after allergenchallenge. The eosinophilia was coincident with the up-regulation inlung epithelial cells of eotaxin, MIP-1 and MCP-1, MCP-3, and MCP-5,which are chemotactic for eosinophils (5). Other evidence for the roleof IL-9 in asthma includes studies of the ability of IL-9 to stimulatemucin secretion by airway epithelial cells (6). Taken together, thesestudies illustrate and support the role of IL-9 in regulating manyclinical hallmarks of asthma and allergic inflammation.

IL-4 and IL-13 are considered pivotal to the development of allergicinflammation and asthma. Studies conducted with animals deficient ineither cytokine, or employing reagents that neutralize either IL-4 orIL-13 function, have elucidated the important role these cytokines playin regulating the primary and secondary immune response leading toairway inflammation and airway hyper-responsiveness (7,8). Cumulatively,these data suggest that IL-4 and IL-13 have overlapping as well asindependent roles in the allergic airways response.

Decreased IL-9, IL-4, and IL-13 activity can decrease Th2 polarizationof the T-cell response, decrease eosinophil survival and neutrophilactivity, and attenuate mucus production by airway epithelial cells.These effects, in turn, can reduce airway hyper-reactivity andremodeling, while increasing gas exchange and clearance, and, therefore,can provide an effective therapeutic modality for several lung diseases,including, for example, asthma, chronic obstructive pulmonary disease(emphysema and chronic bronchitis), and related pulmonary conditions. Assuch, targeting of IL-9, IL-4, and IL-13 can provide a significanttherapeutic benefit as compared to the targeting any of these cytokine,alone. Thus, a need exists for compositions that can modulate the effectof IL-9, IL-4, and IL-13 on cells.

SUMMARY OF THE INVENTION

The present invention relates to a chimeric polypeptide that includes aninterleukin-4 (IL-4) mutein receptor antagonist operatively linked to aninterleukin-9 (IL-9) mutein receptor antagonist. The chimericpolypeptide is characterized, in part, in that it can specificallydisrupt the formation of an IL-4 receptor, an IL-9 receptor, aninterleukin-13 (IL-13) receptor, or a combination thereof, for example,by binding to an IL-4 or IL-9 α polypeptide and reducing or preventingassociation of the α polypeptide with an IL-2 receptor γ polypeptide toform a functional IL-4, IL-9 and/or IL-13 receptor. As such, a chimericpolypeptide of the invention, or a polynucleotide encoding the chimericpolypeptide, is useful, for example, for reducing or inhibiting thespecific binding of IL-4, IL-9 and/or IL-13 to an IL-4, IL-9 and/orIL-13 receptor on a cell. Since the binding of such interleukins totheir specific receptors can be associated with pathologic conditionssuch as asthma and other pulmonary disorders, and with particularcancers such as leukemias, the compositions of the invention can beuseful for ameliorating such pathologic conditions.

A chimeric polypeptide of the invention can contain the IL-4 muteinreceptor antagonist component positioned N-terminal to the IL-9 muteinreceptor antagonist, or can contain the IL-9 mutein receptor antagonistcomponent positioned N-terminal to the IL-4 mutein receptor antagonist.Such chimeric polypeptides are exemplified by the polypeptides shown inSEQ ID NO:2 and SEQ ID NO:4, respectively, which comprise the IL-4mutein (IL-4RA) and IL-9 mutein (IL-9RA) components, and further includea signal peptide sequence, a polyhistidine tag, and a tobacco etch virus(TEV) protease recognition site positioned N-terminal to the IL-4 andIL-9 components. The IL-4 and IL-9 components of the chimericpolypeptide can be directly linked (i.e., the C-terminus of onecomponent directly linked to the N-terminus of the second component), orcan be linked via a spacer molecule that operatively links the twocomponents such that each maintains its respective IL-4, IL-9 and/orIL-13 antagonist activity. In one embodiment, the spacer molecule is apeptide, wherein the chimeric polypeptide comprising the spacer peptidecomprises a fusion protein encoded by a polynucleotide. In anotherembodiment, the spacer molecule allows operative linkage of the IL-4 andIL-9 components via a chemical reaction.

A chimeric polypeptide of the invention can include one or moreadditional operatively linked moieties, which can be positioned at theN-terminus and/or the C-terminus of the chimeric polypeptide, can bepositioned between the IL-4 component and the IL-9 component, and/or canbe bound to an amino acid side chain of one or both of the IL-4 and IL-9components. The moiety can be any type of molecule that can be linked toa polypeptide, including, for example, a peptide, a polynucleotide, asmall organic or inorganic molecule, a carbohydrate, a lipid, or acombination of such molecules. For example, the moiety can be peptidetag (e.g., a polyhistidine tag, a myc tag, a FLAG tag, or a V5 tag), orother tag such as biotin (or avidin or streptavidin). Such a tag canprovide a means to detect the presence of the chimeric polypeptideand/or to purify the chimeric polypeptide from a composition containingthe chimeric polypeptide. A peptide moiety also can be provide arecognition site for a protease (e.g., a TEV protease recognition site).The moiety also can be a detectable label, which can allow forconvenient detection of the chimeric polypeptide, for example, in asample, in which case it can further provide a means to determine theamount of the chimeric polypeptide in the sample; or in a cell, in whichcase it can provide a means to detect binding of the chimericpolypeptide to an IL-4, IL-9 and/or IL-13 receptor, or to an IL-4 and/orIL-9 receptor α polypeptide, expressed by the cell; or in vivo, in whichcase it can provide a means to monitor distribution and/or localizationin a subject to which the chimeric polypeptide was administered. Adetectable label can comprise a radionuclide, a fluorescent moiety, aluminescent moiety, a chemiluminescent moiety, a paramagnetic moiety, anenzyme (or cognate substrate), a receptor (or cognate ligand), or anyother molecule that conveniently can be detected when bound to thechimeric polypeptide.

A chimeric polypeptide of the invention can be present in asubstantially purified (isolated) form, or can be formulated as acomposition that includes one or more carriers. A carrier can be anysubstance in which it is desired to combine the chimeric polypeptide,including, for example, an aqueous or non-aqueous carrier (e.g., abuffer, physiologic saline, or ethanol), or can be stabilizing material(e.g., a carbohydrate) with which the chimeric polypeptide can bemaintained in a dry (e.g., lyophilized) state. In one embodiment, thecomposition includes a physiologically acceptable carrier, apharmaceutically acceptable carrier, or a combination thereof, whereinthe composition can be used when it is desired to contact the chimericpolypeptide with cells and/or to administer the chimeric polypeptide toa subject (e.g., a human subject).

The present invention also relates to a polynucleotide encoding achimeric polypeptide comprising an IL-4 mutein operatively linked to anIL-9 mutein. The polynucleotide can be DNA, RNA or a DNA/RNA hybrid, andcan be single stranded or double stranded. Also provided areoligonucleotides useful for identifying a polynucleotide encoding achimeric polypeptide of the invention, such oligonucleotides beingcharacterized, in part, in that they can hybridize to a region of thepolynucleotide comprising IL-4 encoding sequences and IL-9 encodingsequences, or to a nucleotide sequence complementary thereto.Polynucleotides of the invention are exemplified by a polynucleotidethat encodes a chimeric polypeptide having an amino acid sequence as setforth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:7, and bythe polynucleotides having a nucleotide sequence as set forth in SEQ IDNO:1, SEQ ID NO:3, and SEQ ID NO:6.

In one embodiment, a polynucleotide encoding a chimeric polypeptide ofthe invention is operatively linked to a heterologous nucleic acidmolecule, which can be a functional nucleic acid molecule or can act asa tag to identify the presence of the polynucleotide. As such, aheterologous nucleic acid molecule can comprise or encode atranscriptional regulatory element (e.g., a promoter, an enhancer, or asilencer), a translational regulatory element (e.g., a Kozak consensussequence, a start codon, a ribosome binding sequence, a stop codon, or apoly-adenylation signal), or a combination of transcriptional and/ortranslational regulatory elements. A heterologous nucleic acid moleculealso can encode a peptide, which can function as a tag and/or adetectable label, or can function as a cellular localization domain,which facilitates transport of the expressed chimeric polypeptide intoor out of a particular cellular compartment.

As disclosed herein, a polynucleotide of the invention can be useful fora gene therapy type procedure, wherein the polynucleotide is contactedwith a cell under conditions such that the chimeric polypeptide of theinvention is expressed, wherein the chimeric polypeptide can effect itsantagonist activity. Accordingly, the polynucleotide can be contained ina vector, which can be a cloning vector or an expression vector, andwhich can be a prokaryotic vector, a eukaryotic vector, or a shuttlevector (e.g., a vector that can be passaged in both prokaryotic andeukaryotic cells, or in different types of eukaryotic cells). In oneembodiment, the vector is an expression vector, which contains one ormore regulatory elements that facilitate expression of thepolynucleotide in cells or cell types of interest. The expression vectorcan be a viral vector or a plasmid vector, or can contain components ofboth. Also provided are host cells, which contain a polynucleotideencoding a chimeric polypeptide of the invention, wherein thepolynucleotide can, but need not, be contained in a vector (e.g., anexpression vector). In one aspect, the polynucleotide encoding thechimeric polypeptide is stably integrated into the genome of the hostcell.

The present invention also provides a method of reducing or inhibitingcytokine responsiveness of a cell. Such a method can be performed usingcells in culture (e.g., cells of an established cell line or cellculture, or cells of a subject ex vivo), or the cells can be contactedin vivo (e.g., in an experimental animal system, or in a subjectsuffering from a disorder associated with cellular responsiveness to thecytokine). The method can be practiced, for example, by contacting thecell with a chimeric polypeptide encoding an IL-4 mutein operativelylinked to an IL-9 mutein, or by contacting the cell with apolynucleotide encoding the chimeric polypeptide, under conditionssuitable for expression of the encoded chimeric polypeptide. Cytokine(particularly IL-4, IL-9 and/or IL-13) responsiveness of cells includes,for example, the increase or decrease in cell proliferation mediated bya cytokine, the increase or decrease in protein expression by a cellexposed to the cytokine, including, for example, increased cytokineexpression by cell due to contact with IL-4, IL-9 and/or IL-13.

In various aspects, the cytokine responsiveness of the cell can be IL-4responsiveness, IL-9 responsiveness, or IL-13 responsiveness, or thecell can be a responsive to combination of cytokines (e.g., IL-4 andIL-9, IL-4 and IL-13, or IL-9 and a second or more cytokine(s)). Thecell to be contacted can be any cell that exhibits a response to acytokine, particularly a response mediated by binding of the cytokine toa receptor expressed by the cell. As such, the cell can be, for example,a lymphocyte (e.g., a T lymphocyte or a B lymphocyte), apolymorphonuclear leukocyte (e.g., an eosinophil), a monocyte (e.g., ahistiocyte), or a mast cell. The cell can be a normal cell (e.g., a mastcell) that, due to its cytokine responsiveness, contributes to adisorder (e.g., a pulmonary disorder such as asthma), or can be a cancercell (e.g., a leukemia cell such as an erythroleukemia cell or amegakaryoblastic leukemia cell), wherein the cytokine responsiveness ismanifest by abnormal proliferation.

Accordingly, the present invention also relates to a method ofameliorating a pathologic condition associated with cells expressing atleast one receptor selected from an IL-4 receptor, an IL-9 receptor, oran IL-13 receptor in a subject. The subject can be any subject havingcells that express an IL-4, IL-9 and/or IL-13 receptor, whereinresponsiveness of the cells to a cytokine is associated with apathologic condition. As such, the subject can be a vertebrate subject,including, for example, a mammalian subject, particularly a humansubject suffering from a pathologic condition (e.g., a pulmonarydisorder such as asthma or a cancer such as a leukemia). The method ofameliorating a pathologic condition can be practiced, for example, byadministering to the subject a chimeric polypeptide antagonistcomprising an IL-4 mutein and an IL-9 mutein, or an expressiblepolynucleotide encoding the chimeric polypeptide, in an amountsufficient to reduce or inhibit specific binding of IL-4, IL-9, IL-13,or a combination thereof to the receptor, thereby ameliorating thepathologic condition in the subject. A pathologic condition amenable toamelioration according to the present methods include, for example,pulmonary disorders such as asthma and chronic obstructive pulmonarydisease (e.g., emphysema and chronic bronchitis), and allergicinflammatory responses. Additional pathologic conditions amenable totreatment according to the present methods include cancers such asleukemias and lymphomas (e.g., non-Hodgkin's lymphoma).

The present invention also relates to a method of making a chimericpolypeptide, which comprises an IL-4 mutein receptor antagonistoperatively linked to an IL-9 mutein receptor antagonist. Such a methodconveniently can be performed by expressing a polynucleotide encodingthe chimeric polypeptide. For example, the polynucleotide, which can,but need not, be contained in a vector, can be contained in a host cell,which can be cultured under conditions whereby the polynucleotide isexpressed and the chimeric polypeptide is produced. Accordingly, theinvention provides an IL-4 mutein receptor antagonist operatively linkedto an IL-9 mutein receptor antagonist, produced by such a method.Further, the method can include a step of purifying the chimericpolypeptide. As such, the invention also provides a purified IL-4 muteinreceptor antagonist operatively linked to an IL-9 mutein receptorantagonist produced by such a method.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that a chimericpolypeptide comprising an interleukin-4 (IL-4) mutein (IL-4RA) and aninterleukin-9 (IL-9) mutein (IL-9RA) acts as an antagonist of IL-4, IL-9and IL-13 mediated responsiveness of cells involved in immune andinflammatory responses. Accordingly, in one embodiment, the inventionprovides a chimeric polypeptide that includes an IL-4 mutein receptorantagonist operatively linked to an IL-9 mutein receptor antagonist,which can specifically disrupt the formation of an IL-4 receptor, anIL-9 receptor, an interleukin-13 (IL-13) receptor. In anotherembodiment, the chimeric polypeptide comprises an IL-4RA or an IL-9RAoperatively linked to an IL-9 or an IL-4 component, respectively. Alsoprovided are polynucleotides encoding such chimeric mutein antagonists,as well as methods of using the chimeric polypeptide, or encodingpolynucleotide, to reduce or inhibit the specific binding of IL-4, IL-9and/or IL-13 to an IL-4, IL-9 and/or IL-13 receptor on a cell.

Chimeric recombinant muteins have been developed from a human IL-9mutein (IL-9RA), which inhibits IL-9-induced cell proliferation, andhuman IL-4 mutein, which is mutated in two positions of its amino acidsequence (IL-4RA). IL-9RA and IL-4RA were designed to bind to theirrespective IL-9 and IL-4 receptor alpha chains, thereby competitivelyinhibiting binding of the wild-type cytokines to the receptor and/orreducing or inhibiting the association of the IL-4 and/or IL-9 αpolypeptide with an IL-2 receptor γ polypeptide (thus preventingassembly of a functional IL-4, IL-9, and/or IL-13 receptor). Wild-typeIL-9 signals through a two subunit receptor that consists of IL-9Rα andthe common γ chain, which is a shared component of the receptorcomplexes for IL-2, IL-4, IL-7, IL-13, IL-15, and possibly others. Incomparison, IL-4 signals through the IL-4Rα and the common γ chains. TheIL-4Rα chain also is a functional signaling component of the IL-13receptor complex.

Various disorders, including, for example, pulmonary disorders such asasthma, are associated with cell responsiveness to cytokines. Asdisclosed herein, IL-9, IL-4, and IL-13-mediated cellular activity,including, for example, cytokine induced cell proliferation, can beattenuated by inhibiting cytokine signaling through their respectivecognate cell-surface receptors using operatively linked forms of IL-9RAand IL-4RA chimeric polypeptide antagonists (see Example 1). The IL-9RAand IL-4RA chimeric mutein antagonists competitively inhibit theabilities of wild type IL-9, IL-4, and IL-13 to signal by blockingbinding of the cytokines to their respective receptors. Accordingly, thepresent invention provides chimeric polypeptides, including, forexample, fusion proteins, that comprise an IL-9 mutein and an IL-4mutein, polynucleotides encoding such IL-9 and IL-4 chimeric antagonistmuteins, compositions comprising the polypeptides and/orpolynucleotides, and methods of using the compositions, for example, tomodulate cytokine-mediated responsiveness of a cell, and to ameliorate apathologic condition associated with cytokine-mediated responsiveness.

The present invention provides reagents and methods of inhibiting immuneresponses mediated by IL-4, IL-9 and IL-13. For example, a purifiedprotein preparation comprising a chimeric (fusion) protein, whichincludes of a modified IL-4 mutein receptor antagonist operativelylinked to an IL-9 mutein receptor antagonist, is provided. The IL-4RAcomponent of the fusion protein can be located N-terminal to the IL-9RAcomponent (“IL-4RA/IL-9RA”; see, e.g., SEQ ID NO:2), or the IL-9RAcomponent of the fusion protein can be located N-terminal to the IL-4RA(“IL-9RA/IL-4RA”; see, e.g., SEQ ID NO:4; see, also, SEQ ID NOS:5 and7). As used herein, the designation of a chimeric polypeptide indicatesthe N-terminal to C-terminal orientation of the IL-4 and IL-9 components(e.g., “IL-4RA/IL-9RA” indicates that the IL-4 mutein is N-terminal tothe IL-9 mutein); the designation “RN” indicates that component (e.g.,IL-4) is a mutein (i.e., IL-4RA) that can act as a cytokine receptorantagonist. As disclosed herein, one or, preferably, both of the IL-4and IL-9 components of the receptor antagonist is a mutein. As usedherein, reference to “an IL-4RA and IL-9RA chimeric polypeptide” or “anIL-9RA and IL-4RA chimeric antagonist” or “an IL-4 and IL-9 muteinfusion protein” or the like means that the IL-4 component can beN-terminal or C-terminal to the IL-9 component in the chimeric muteinantagonist.

The term “operatively linked” is used herein to refer to two or moremolecules that share a covalent or non-covalent interaction, whereineach molecule maintains some or all of the function that the moleculeexhibits alone. The two or more molecules, which can be peptides(polypeptides), polynucleotides, or other molecules, can be linkeddirectly to each other, or can be operatively linked via a linker(spacer) molecule. For example, a polynucleotide encoding a firstpeptide, e.g., IL-4RA, can be operatively linked to a polynucleotideencoding a heterologous peptide, e.g., IL-9RA, such that the nucleotidesequences are in frame. Upon expression of such a recombinantpolynucleotide, the peptides are expressed as a fusion protein, whereinthe IL-4 and IL-9 components are operatively linked, and the IL-4RAcomponent of the fusion protein can specifically bind at least to anIL-4 receptor, particularly to an IL-4 receptor α polypeptide, and theIL-9RA component can specifically bind at least to an IL-9 receptor,particularly to an IL-9 receptor α polypeptide.

The term “fusion protein” or “chimeric polypeptide” or the like is usedherein to mean two more peptides that are operatively linked. A fusionprotein can be obtained, for example, by expression a recombinantpolynucleotide encoding the fusion protein, or a chimeric protein can beobtained by chemically linking a first peptide (e.g., IL-4 or an IL-4RA)to a second (or other) peptide (e.g., IL-9 or an IL-9 mutein), or bychemically synthesizing the entire protein comprising the peptidecomponents. A chimeric protein of the invention also can be obtainedusing a combination of such methods, including, for example, byexpressing a fusion polypeptide comprising IL-4RA operatively linked toIL-9RA, then further chemically linking a peptide tag (e.g., apolyhistidine tag) to the fusion protein.

The term “operatively linked” also is used to refer to a recombinantpolynucleotide containing two or more linked polynucleotides, each ofwhich maintains a function characteristic of the polynucleotide. Forexample, a recombinant polynucleotide can comprise an IL-4RA codingsequence operatively linked to an IL-9RA coding sequences, wherein thecoding sequences are in-frame and can be expressed as a fusion proteinhaving IL-4RA activity and IL-9RA activity. A recombinant polynucleotidealso can include, for example, a transcriptional promoter (or otherregulatory element) operatively linked to a coding sequence, wherein thetranscriptional promoter can drive expression of the coding sequenceunder the appropriate conditions for expression of a polynucleotide fromthe promoter. As such, a first polynucleotide coding sequence can beoperatively linked to a second (or more) coding sequence such that achimeric polypeptide can be expressed from the operatively linked codingsequences.

The term “peptide” or “polypeptide” or “protein” is used broadly hereinto mean two or more amino acids linked by a peptide bond. Generally, apeptide of the invention comprises a chimeric polypeptide, althoughpeptide fragments comprising the linked regions of two peptides arecontemplated. A peptide generally contains at least about six aminoacids, usually contains about ten amino acids, and can contain fifteenor more amino acids, particularly twenty or more amino acids. It shouldbe recognized that the term “peptide” is not used herein to suggest aparticular size or number of amino acids comprising the molecule, andthat a peptide of the invention can contain up to several amino acidresidues or more. As used herein, the term “substantially purified” or“substantially pure” or “isolated” means that the molecule beingreferred to, for example, a peptide or a polynucleotide, is in a formthat is relatively free of proteins, nucleic acids, lipids,carbohydrates or other materials with which it is naturally associated(i.e., in nature). Generally, a substantially pure peptide,polynucleotide, or other molecule constitutes at least twenty percent ofa sample, generally constitutes at least about fifty percent of asample, usually constitutes at least about eighty percent of a sample,and particularly constitutes about ninety percent or ninety-five percentor more of a sample.

As disclosed herein, the inclusion of a polyhistidine tag operativelylinked to an IL-4RA and IL-9RA chimeric polypeptide provided a means tosubstantially purify the expressed chimeric polypeptide. Further, theinclusion of a TEV protease cleavage site between the tag and the IL-4RAand IL-9RA components provided a means to cleave the tag from thechimeric polypeptide antagonist (see, e.g., SEQ ID NOS:2 and 4). Adetermination that a peptide or a polynucleotide of the invention issubstantially pure can be made using well known methods, for example, byperforming electrophoresis and identifying the particular molecule as arelatively discrete band. A substantially pure polynucleotide, forexample, can be obtained by cloning the polynucleotide, or by chemicalor enzymatic synthesis. A substantially pure peptide can be obtained,for example, by a method of chemical synthesis, or using methods ofprotein purification, followed by proteolysis and, if desired, furtherpurification by chromatographic or electrophoretic methods.

A chimeric polypeptide of the invention can correspond to an amino acidsequence of an IL-4 mutein and/or IL-9 mutein, or can vary from themutein sequence, for example, by containing one or more D-amino acids inplace of a corresponding L-amino acid; or by containing one or moreamino acid analogs, for example, an amino acid that has been derivatizedor otherwise modified at its reactive side chain. Similarly, one or morepeptide bonds in the peptide can be modified. In addition, a reactivegroup at the amino terminus or the carboxy terminus or both can bemodified. As such, the chimeric polypeptide antagonists can be modified,for example, to have improved stability to a protease, an oxidizingagent or other reactive material the peptide may encounter in abiological environment, and, therefore, can be particularly useful inperforming a method of the invention. Of course, the peptides can bemodified to have decreased stability in a biological environment suchthat the period of time the peptide is active in the environment isreduced.

The sequence of a chimeric polypeptide of the invention also can bemodified in comparison to the corresponding sequence in an IL-4RA and/orIL-9RA component by incorporating a conservative amino acid substitutionfor one or a few amino acids in the peptide component. Conservativeamino acid substitutions include the replacement of one amino acidresidue with another amino acid residue having relatively the samechemical characteristics, for example, the substitution of onehydrophobic residue such as isoleucine, valine, leucine or methioninefor another, or the substitution of one polar residue for another, forexample, substitution of arginine for lysine; or of glutamic foraspartic acid; or of glutamine for asparagine; or the like, providedthat the substitution does not result in a loss of IL-4, IL-9, and/orIL-13 antagonist activity of the chimeric polypeptide antagonist.

Modified IL-4 mutein receptor antagonists are described, for example, inU.S. Pat. Nos. 5,723,118 and 6,130,318, each of which is incorporatedherein by reference). Modified IL-9 mutein receptor antagonists aredescribed, for example, in International Application NoPCT/US2004/015168, which is incorporated herein by reference. Asdisclosed, mutations can be introduced into the D helix, including, forexample, a substitution of lysine to glutamic acid at position 126(K126E) and a substitution of glutamine for lysine at position 133(Q133K) in IL-9 (or any other mutation(s) as disclosed in Int'l Appl.No. PCT/US2004/015168), to generate a double mutein IL9 and IL4 chimericreceptor antagonist. Methods for determining that a substitution at oneor more positions of IL-4 and/or IL-9 results in a mutein antagonistuseful for preparing a chimeric polypeptide antagonist of the inventionare disclosed herein (see Example 1) and otherwise known in the art(see, e.g., U.S. Pat. Nos. 5,723,118 and 6,130,318, and Int'l. Appl. No.PCT/US2004/015168). For example, the capacity of the IL-4RA and IL-9RAmutein fusion proteins to reduce or inhibit the proliferative responseof immune cells was assessed using proliferative assays as outlined inExample 1, and expressed as an “Inhibitory Concentration 50%” (IC₅₀).Generally, a chimeric polypeptide antagonist of the present inventioncan prevent the assembly of human IL-4 and IL-9 receptors and neutralizetheir capacity to prevent immune cell proliferation with a preferredIC₅₀ ranging from about 0.1 nM to 10 μM, including, for example, about1.0 nM to 100 nM, or about 0.5 nM to 1 μM.

Also provided are polynucleotides encoding an IL-4RA and IL-9RA chimericantagonist mutein, including, for example, a polynucleotide encoding anIL-4RA/IL-9RA fusion protein (see, e.g., SEQ ID NO:1) and apolynucleotide encoding an IL-9RA/IL-4RA fusion protein (see, e.g., SEQID NO:3 and SEQ ID NO:6). Accordingly, the invention further providesvectors (e.g., expression vectors) containing a polynucleotide encodingan IL-4 and IL-9 chimeric antagonist mutein, as well as host cellscontaining such polynucleotides and/or vectors. In addition, theinvention provide methods of making an IL-4 and IL-9 mutein fusionprotein by culturing such a host cell under conditions suitable forexpression of the antagonist. Such a method can further includepurifying the antagonist from the host cell culture, thus providing ameans to obtain a substantially purified antagonist.

The term “polynucleotide” is used broadly herein to mean a sequence oftwo or more deoxyribonucleotides or ribonucleotides that are linkedtogether by a phosphodiester bond. As such, the term “polynucleotide”includes RNA and DNA, which can be a gene or a portion thereof, a cDNA,a synthetic polydeoxyribonucleic acid sequence, or the like, and can besingle stranded or double stranded, as well as a DNA/RNA hybrid.Furthermore, the term “polynucleotide” as used herein includes naturallyoccurring nucleic acid molecules, which can be isolated from a cell, aswell as synthetic molecules, which can be prepared, for example, bymethods of chemical synthesis or by enzymatic methods such as by thepolymerase chain reaction (PCR). In various embodiments, apolynucleotide of the invention can contain nucleoside or nucleotideanalogs, or a backbone bond other than a phosphodiester bond (seeabove).

In general, the nucleotides comprising a polynucleotide are naturallyoccurring deoxyribonucleotides, such as adenine, cytosine, guanine orthymine linked to 2′-deoxyribose, or ribonucleotides such as adenine,cytosine, guanine or uracil linked to ribose. However, a polynucleotidealso can contain nucleotide analogs, including non-naturally occurringsynthetic nucleotides or modified naturally occurring nucleotides. Suchnucleotide analogs are well known in the art and commercially available,as are polynucleotides containing such nucleotide analogs (Lin et al.,Nucl. Acids Res. 22:5220-5234 (1994); Jellinek et al., Biochemistry34:11363-11372 (1995); Pagratis et al., Nature Biotechnol. 15:68-73(1997), each of which is incorporated herein by reference).

The covalent bond linking the nucleotides of a polynucleotide generallyis a phosphodiester bond. However, the covalent bond also can be any ofnumerous other bonds, including a thiodiester bond, a phosphorothioatebond, a peptide-like bond or any other bond known to those in the art asuseful for linking nucleotides to produce synthetic polynucleotides(see, for example, Tam et al., Nucl. Acids Res. 22:977-986 (1994); Eckerand Crooke, BioTechnology 13:351360 (1995), each of which isincorporated herein by reference). The incorporation of non-naturallyoccurring nucleotide analogs or bonds linking the nucleotides or analogscan be particularly useful where the polynucleotide is to be exposed toan environment that can contain a nucleolytic activity, including, forexample, a tissue culture medium or upon administration to a livingsubject, since the modified polynucleotides can be less susceptible todegradation.

A polynucleotide comprising naturally occurring nucleotides andphosphodiester bonds can be chemically synthesized or can be producedusing recombinant DNA methods, using an appropriate polynucleotide as atemplate. In comparison, a polynucleotide comprising nucleotide analogsor covalent bonds other than phosphodiester bonds generally will bechemically synthesized, although an enzyme such as T7 polymerase canincorporate certain types of nucleotide analogs into a polynucleotideand, therefore, can be used to produce such a polynucleotiderecombinantly from an appropriate template (Jellinek et al., supra,1995).

Where a polynucleotide encodes a peptide, for example, a chimericpolypeptide comprising an IL-4 and/or IL-9 mutein, the coding sequencecan be contained in a vector, wherein it can be operatively linked toappropriate regulatory elements, including, if desired, a tissuespecific promoter or enhancer. The encoded peptide can be furtheroperatively linked, for example, to peptide tag such as a polyhistidine(e.g., His6) tag or the like, which can facilitate identification ofexpression of the chimeric polypeptide in a sample (e.g., in a targetcell). A polyhistidine tag peptide can be detected using a divalentcation such as nickel ion, cobalt ion, or the like. Additional peptidetags include, for example, a FLAG epitope, which can be detected usingan anti-FLAG antibody (see, for example, Hopp et al., BioTechnology6:1204, 1988; U.S. Pat. No. 5,011,912, each of which is incorporatedherein by reference); a c-myc epitope, which can be detected using anantibody specific for the epitope; biotin, which can be detected usingstreptavidin or avidin; and glutathione S-transferase, which can bedetected using glutathione. Such tags, which can be operatively linkedto the N-terminus, C-terminus, or both of a chimeric IL4 and/or IL-9mutein, can provide the additional advantage that they can facilitateisolation of the operatively linked chimeric polypeptide, for example,where it is desired to obtain the chimeric polypeptide in asubstantially purified form. As such, the polynucleotides of theinvention can provide a convenient means to obtain desired amounts ofthe chimeric polypeptides (e.g., sufficient quantities for therapeuticuse).

The polynucleotides of the invention, which can be in a vector, can becontained in a host cell, or can be isolated free of other cellularcomponents such as membrane components, proteins, and lipids. Isolatedpolynucleotides can be obtained, for example, from such host cells usingstandard nucleic acid purification techniques, or can be synthesizedusing an amplification technique (e.g., PCR) or using an automaticsynthesizer. For example, restriction enzymes and probes can be used toidentify and isolate a polynucleotide encoding an IL-4 and IL-9 chimericmutein antagonist polypeptide.

A polynucleotide encoding an IL-4 and IL-9 chimeric mutein antagonistcan be contained in a vector, which can facilitate manipulation of thepolynucleotide, including introduction of the polynucleotide into atarget cell. The vector can be a cloning vector, which is useful formaintaining the polynucleotide, or can be an expression vector, whichcontains, in addition to the polynucleotide, regulatory elements usefulfor expressing the encoded chimeric antagonist polypeptide. Anexpression vector can contain the expression elements necessary toachieve, for example, sustained transcription of the encodingpolynucleotide, or the regulatory elements can be operatively linked tothe polynucleotide prior to its being cloned into the vector.

An expression vector (or the polynucleotide) generally contains orencodes a promoter sequence, which can provide constitutive or, ifdesired, inducible or tissue specific or developmental stage specificexpression of the encoding polynucleotide, a poly-A recognitionsequence, and a ribosome recognition site or internal ribosome entrysite, or other regulatory elements such as an enhancer, which can betissue specific. The vector also can contain elements required forreplication in a prokaryotic (e.g., bacterial) or eukaryotic (e.g.,insect, yeast (e.g., Pichia) and/or mammalian) host system or both, asdesired. Such vectors, which include plasmid vectors and viral vectorssuch as bacteriophage, baculovirus, retrovirus, lentivirus, adenovirus,vaccinia virus, semliki forest virus and adeno-associated virus vectors,are well known and can be purchased from a commercial source (Promega,Madison Wis.; Stratagene, La Jolla Calif.; GIBCO/BRL, Gaithersburg Md.;Invitrogen Corp., Carlsbad Calif.) or can be constructed by one skilledin the art (see, for example, Meth. Enzymol., Vol. 185, Goeddel, ed.(Academic Press, Inc., 1990); Jolly, Canc. Gene Ther 1:51-64, 1994;Flotte, J. Bioenerg. Biomemb. 25:37-42, 1993; Kirshenbaum et al., J.Cln. Invest. 92:381-387, 1993, each of which is incorporated herein byreference).

An inducible promoter such as the tetracycline (tet) promoter can beparticularly useful for driving expression of a polynucleotide encodingan IL-4 and IL-9 mutein fusion protein. Upon administration oftetracycline, or a tetracycline analog, to a subject containing apolynucleotide operatively linked to a tet inducible promoter,expression of the encoded chimeric polypeptide is induced, whereby thechimeric polypeptide can effect its IL-4, IL-9 and/or IL-13 antagonistactivity. Alternatively, or in addition, the polynucleotide can beoperatively linked to tissue specific regulatory element, for example, alung epithelial cell specific regulatory element, such that expressionof an encoded peptide is restricted to the lung cells in an individual,or to lung cells in a mixed population of cells in culture, for example,an organ culture. In such a case, the encoded chimeric polypeptide canfurther contain a signal peptide, such that the chimeric polypeptide isexported extracellularly, where it can effect its antagonist activity oncytokine responsive cells.

Viral expression vectors can be particularly useful for introducing apolynucleotide into a cell, particularly a cell in a subject. Viralvectors provide the advantage that they can infect host cells withrelatively high efficiency and can infect specific cell types. Forexample, a polynucleotide encoding a chimeric polypeptide antagonist ofthe invention can be cloned into a baculovirus vector, which then can beused to infect an insect host cell, thereby providing a means to producelarge amounts of the encoded chimeric polypeptide. The viral vector alsocan be derived from a virus that infects cells of an organism ofinterest, for example, vertebrate host cells such as mammalian, avian orpiscine host cells. Viral vectors can be particularly useful forintroducing a polynucleotide useful in performing a method of theinvention into a target cell. Viral vectors have been developed for usein particular host systems, particularly mammalian systems and include,for example, retroviral vectors, other lentivirus vectors such as thosebased on the human immunodeficiency virus (HIV), adenovirus vectors,adeno-associated virus vectors, herpesvirus vectors, vaccinia virusvectors, and the like (see Miller and Rosman, BioTechniques 7:980-990,1992; Anderson et al., Nature 392:25-30 Suppl., 1998; Verma and Somia,Nature 389:239-242, 1997; Wilson, New Engl. J. Med. 334:1185-1187(1996), each of which is incorporated herein by reference).

A polynucleotide, which can be contained in a vector, can be introducedinto a cell by any of a variety of methods known in the art (Sambrook etal., “Molecular Cloning:

A laboratory manual” (Cold Spring Harbor Laboratory Press 1989); Ausubelet al., “Current Protocols in Molecular Biology”, John Wiley and Sons,Baltimore, Md. (1987, and supplements through 1995), each of which isincorporated herein by reference). Such methods include, for example,transfection, lipofection, microinjection, electroporation and, withviral vectors, infection; and can include the use of liposomes,microemulsions or the like, which can facilitate introduction of thepolynucleotide into the cell and can protect the polynucleotide fromdegradation prior to its introduction into the cell. The selection of aparticular method will depend, for example, on the cell into which thepolynucleotide is to be introduced, as well as whether the cell isisolated in culture, or is in a tissue or organ in culture or in situ.

Introduction of a polynucleotide into a cell by infection with a viralvector is particularly advantageous in that it can efficiently introducethe nucleic acid molecule into a cell ex vivo or in vivo (see, forexample, U.S. Pat. No. 5,399,346, which is incorporated herein byreference). Moreover, viruses are very specialized and can be selectedas vectors based on an ability to infect and propagate in one or a fewspecific cell types. Thus, their natural specificity can be used totarget the nucleic acid molecule contained in the vector to specificcell types. As such, a vector based on an HIV can be used to infect Tcells, a vector based on an adenovirus can be used, for example, toinfect respiratory epithelial cells, a vector based on a herpesvirus canbe used to infect neuronal cells, and the like. Other vectors, such asadeno-associated viruses can have greater host cell range and,therefore, can be used to infect various cell types, although viral ornon-viral vectors also can be modified with specific receptors orligands to alter target specificity through receptor mediated events.

A chimeric protein of the invention can be used to modulate theresponsiveness of a cell to a cytokine, particularly to IL-4, IL-9,and/or IL-13, depending on the responsiveness of the cell. In oneembodiment, the chimeric protein can inhibit the proliferative responseof erythroleukemia cells (e.g., TF-1 cells) to IL-4, or to IL-13, or toboth IL-4 and IL-13, with an IC₅₀ (concentration that inhibitsproliferation of 50% of the cells) at a concentration of about 0.1 nM toabout 10 μM (e.g., about 0.5 nM to about 1 μM, or about 1.0 nM to about100 nM). In another embodiment, the chimeric protein can inhibit theproliferative response of megakaryoblastic leukemia cells (e.g., Mo7ecells) to IL-4, or to IL-9, or to both IL-4 and IL-9, with an IC₅₀ ofabout 0.1 nM to about 10 μM (e.g., about 0.5 nM to about 1 μM, or about1.0 nM to about 100 nM). In still another embodiment, the chimericprotein can inhibit the proliferative response of B cells, or of Tcells, or of both B cells and T cells (e.g., human B cells and/or Tcells) to IL-4 with an IC₅₀ of about 0.1 nM to about 10 μM (e.g., about0.5 nM to about 1 μM, or about 1.0 nM to about 100 nM).

In various aspects, the method of modulating the responsiveness of cellsto a cytokine provides methods of preventing a pathologic condition andtherapeutic methods for ameliorating a pathologic condition associatedwith such cellular responsiveness. The pathologic conditions can bemanifest as an aberrant immune response and/or an aberrant inflammatoryresponse, including, for example, an allergic inflammatory reaction,asthma, and chronic obstructive pulmonary disorders (e.g., emphysema andbronchitis). Accordingly, the invention provides a method of treating adisorder in which IL-4 and/or IL-9 receptors are expressed in cellsassociated with the disorder, particularly disorders of vertebrates,including mammals (e.g., humans). In one embodiment, the method isperformed by administering a chimeric protein that antagonizes IL-4,IL-9 and/or IL-13 mediated activity to a subject to be treated (e.g., ahuman), wherein the chimeric protein is administered in an amountsufficient to antagonize IL-4, IL-9 and/or IL-13-mediated activity,thereby ameliorating the pathologic condition. In another embodiment,the method is performed by administering an expressible polynucleotideencoding a chimeric protein that antagonizes IL-4, IL-9 and/or IL-13mediated activity to a subject to be treated (e.g., a human), whereinthe polynucleotide, which can be contained in a vector, is administeredsuch that it can enter cells in which the chimeric polypeptide can beexpressed and released from the cells in an amount sufficient toantagonize IL-4, IL-9 and/or IL-13-mediated activity, therebyameliorating the pathologic condition

The present invention also provides compositions, includingpharmaceutical compositions, which can be administered to a subjectsuffering from such a pathologic condition (e.g., a pulmonary disorder)associated with cytokine expression and/or cellular responsiveness tocytokines, particularly IL-4, IL-9, and/or IL-13. Such a composition ofthe invention includes a chimeric protein comprising, for example, anIL-4RA operatively linked to an IL-9RA, wherein the chimeric protein canspecifically bind to an IL-4 receptor α polypeptide and/or an IL-9receptor α polypeptide (thereby disrupting the assembly of a human IL-4,IL-9, and/or IL-13 receptor), or a polynucleotide encoding the chimericprotein, and a pharmaceutically acceptable carrier suitable foradministration to the intended subject. As used herein, the term“specifically binds” means that two molecules form a complex that isrelatively stable under physiologic conditions. The term is used hereinin reference to an interleukin and its receptor (e.g., IL-4 and an IL-4receptor), including to an interleukin mutein (e.g., IL-4RA) and acognate interleukin receptor or component thereof (e.g., an IL-4receptor α polypeptide). Specific binding can be characterized by adissociation constant of at least about 1×10⁻⁶ M, generally at leastabout 1×10⁻⁷ M, usually at least about 1×10⁻⁸ M, and particularly atleast about 1×10⁻⁹ M or 1×10⁻¹⁰ M or greater. Specific binding generallyis stable under physiological conditions, including, for example,conditions that occur in a living individual such as a human or othervertebrate or invertebrate, as well as conditions that occur in a cellculture such as used for maintaining mammalian cells or cells fromanother vertebrate organism or an invertebrate organism. Methods fordetermining whether two molecules specifically bind are well known andinclude, for example, equilibrium dialysis, surface plasmon resonance,two hybrid assays, and the like. Specific binding of a chimeric proteincomprising, for example, an IL-4RA operatively linked to an IL-9RAchimeric polypeptide can be identified by detecting disruption ofbinding of an interleukin (e.g., IL-4) with its receptor, by detecting areduction or inhibition of binding of an interleukin with a cognatereceptor, and/or by detecting a reduction or inhibition of assembly ofIL-4 and/or IL-9 α polypeptide with an IL-2 γ polypeptide to form afunctional interleukin receptor.

As disclosed herein, composition of the invention can have use intreating a disorder associated with cytokine responsiveness of cells,including, for example, ameliorating pulmonary disorders such as asthmaand bronchitis, as well as cancers such as leukemias and lymphomas. Assuch, the chimeric polypeptides and encoding polynucleotides are usefulas medicaments for use in treating a subject suffering from apathological condition associated with cytokine (e.g., IL-4, IL-9,and/or IL-13) induced cell responsiveness.

Pharmaceutically acceptable carriers are well known in the art andinclude, for example, aqueous solutions such as water or physiologicallybuffered saline or other solvents or vehicles such as glycols, glycerol,oils such as olive oil or injectable organic esters. A pharmaceuticallyacceptable carrier can contain physiologically acceptable compounds thatact, for example, to stabilize or to increase the absorption of theconjugate. Such physiologically acceptable compounds include, forexample, carbohydrates, such as glucose, sucrose or dextrans,antioxidants, such as ascorbic acid or glutathione, chelating agents,low molecular weight proteins or other stabilizers or excipients. Oneskilled in the art would know that the choice of a pharmaceuticallyacceptable carrier, including a physiologically acceptable compound,depends, for example, on the physico-chemical characteristics of thetherapeutic agent and on the route of administration of the composition,which can be, for example, orally, via inhalation, or parenterally suchas intravenously, and by injection, intubation, or other such methodknown in the art. In addition to an IL-4 and IL-9 chimeric muteinpolypeptide, the pharmaceutical composition also can contain a secondreagent such as a diagnostic reagent, nutritional substance, toxin, ortherapeutic agent, for example, a cancer chemotherapeutic agent.

The chimeric polypeptide can be incorporated within an encapsulatingmaterial such as into an oil-in-water emulsion, a microemulsion,micelle, mixed micelle, liposome, microsphere or other polymer matrix(see, for example, Gregoriadis, “Liposome Technology”, Vol. 1 (CRCPress, Boca Raton, Fla. 1984); Fraley, et al., Trends Biochem. Sc., 6:77(1981), each of which is incorporated herein by reference). Liposomes,for example, which consist of phospholipids or other lipids, arenontoxic, physiologically acceptable and metabolizable carriers that arerelatively simple to make and administer. “Stealth” liposomes (see, forexample, U.S. Pat. Nos. 5,882,679; 5,395,619; and 5,225,212, each ofwhich is incorporated herein by reference) are an example of suchencapsulating materials particularly useful for preparing apharmaceutical composition useful for practicing a method of theinvention, and other “masked” liposomes similarly can be used, suchliposomes extending the time that the therapeutic agent remain in thecirculation. Cationic liposomes, for example, also can be modified withspecific receptors or ligands (Morishita et al., J. Clin. Invest.,91:2580-2585 (1993), which is incorporated herein by reference). Inaddition, a polynucleotide agent can be introduced into a cell using,for example, adenovirus-polylysine DNA complexes (see, for example,Michael et al., J. Biol. Chem. 268:6866-6869 (1993), which isincorporated herein by reference).

The route of administration of a pharmaceutical composition containing achimeric polypeptide (or encoding polynucleotide) of the invention willdepend, in part, on the chemical structure of the molecule. Polypeptidesand polynucleotides, for example, are not particularly useful whenadministered orally because they can be degraded in the digestive tract.However, methods for chemically modifying polypeptides, for example, torender them less susceptible to degradation by endogenous proteases ormore absorbable through the alimentary tract are well known (see, forexample, Blondelle et al., supra, 1995; Ecker and Crook, supra, 1995).In addition, a chimeric polypeptide can be prepared using D-amino acids,or can contain one or more domains based on peptidomimetics, which areorganic molecules that mimic the structure of peptide domain; or basedon a peptoid such as a vinylogous peptoid.

A pharmaceutical composition as disclosed herein can be administered toan individual by various routes including, for example, via inhalation,orally or parenterally, such as intravenously, intramuscularly,subcutaneously, intraorbitally, intracapsularly, intraperitoneally,intratracheally, intrarectally, intracisternally or by passive orfacilitated absorption through the skin using, for example, a skin patchor transdermal iontophoresis, respectively. Furthermore, thepharmaceutical composition can be administered by injection, intubation,orally or topically, the latter of which can be passive, for example, bydirect application of an ointment, or active, for example, using a nasalspray or inhalant, in which case one component of the composition is anappropriate propellant. A pharmaceutical composition also can beadministered to the site of a pathologic condition, for example,intravenously or intra-arterially into a blood vessel supplying a tumor,or via inhalation for treatment of a pulmonary disorder.

The total amount of an agent to be administered in practicing a methodof the invention can be administered to a subject as a single dose,either as a bolus or by infusion over a relatively short period of time,or can be administered using a fractionated treatment protocol, in whichmultiple doses are administered over a prolonged period of time. Oneskilled in the art would know that the amount of the pharmaceuticalcomposition to treat a pathologic condition in a subject depends on manyfactors including the age and general health of the subject as well asthe route of administration and the number of treatments to beadministered. In view of these factors, the skilled artisan would adjustthe particular dose as necessary. In general, the formulation of thepharmaceutical composition and the routes and frequency ofadministration are determined, initially, using Phase I and Phase IIclinical trials.

The pharmaceutical composition can be formulated for oral formulation,such as a tablet, or a solution or suspension form; or can comprise anadmixture with an organic or inorganic carrier or excipient suitable forenteral or parenteral applications, and can be compounded, for example,with the usual non-toxic, pharmaceutically acceptable carriers fortablets, pellets, capsules, suppositories, solutions, emulsions,suspensions, or other form suitable for use. The carriers, in additionto those disclosed above, can include glucose, lactose, mannose, gumacacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc,corn starch, keratin, colloidal silica, potato starch, urea, mediumchain length triglycerides, dextrans, and other carriers suitable foruse in manufacturing preparations, in solid, semisolid, or liquid form.In addition auxiliary, stabilizing, thickening or coloring agents andperfumes can be used, for example a stabilizing dry agent such astriulose (see, for example, U.S. Pat. No. 5,314,695).

As disclosed herein, the fusion protein antagonists of the invention canbe provided in a pharmaceutical composition comprising apharmaceutically acceptable carrier. The pharmaceutically acceptablecarrier preferably is non-pyrogenic. The compositions can beadministered alone or in combination with at least one other agent(e.g., a stabilizing compound), and can be administered in any sterile,biocompatible pharmaceutical carrier, including, for example, saline,buffered saline, dextrose, and water. A variety of aqueous carriers maybe employed, e.g., 0.4% saline, 0.3% glycine, and the like. Thesesolutions are sterile and generally free of particulate matter. Thesesolutions can be sterilized by conventional, well known sterilizationtechniques (e.g., filtration). The compositions also can containpharmaceutically acceptable auxiliary substances to approximatephysiological conditions such as pH and buffering capacity. Theconcentration of the chimeric polypeptide antagonist in thepharmaceutical formulation can vary as desired, including, for example,from less than about 0.5% (e.g., 0.05%, 0.1%, or 0.25%) to about 20%(e.g., 1%, 2%, 3%, 4%, 5%, 10%, or 15%) by weight, and can be selectedbased, for example, on fluid volume, viscosities, or other parameter asis known in the art for a particular mode of administration selected. Ifdesired, two or more different cytokine antagonists can be included in aformulation, including, for example, chimeric polypeptide antagonistshaving different dissociation constants (K_(d)) for IL-4 and/or IL-9receptor binding.

The compositions can be administered to a patient alone, or incombination with other agents, drugs or hormones. In addition to theactive ingredients, the pharmaceutical compositions can contain suitablepharmaceutically acceptable carriers comprising excipients andauxiliaries that facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Pharmaceuticalcompositions of the invention can be administered by any number ofroutes including, for example, oral, intravenous, intramuscular,intra-arterial, intramedullary, intrathecal, intraventricular,transdermal, subcutaneous, intraperitoneal, intratracheal, intranasal,parenteral, topical, sublingual, or rectally. Upon preparation, apharmaceutical composition can be placed in an appropriate container andlabeled for treatment of an indicated condition. Such labeling caninclude, for example, information relevant to the amount, frequency,and/or method of administration.

The present invention also provides methods of ameliorating a pathologiccondition associated with cells expressing at least one receptorselected from an IL-4 receptor, an IL-9 receptor, or an IL-13 receptorin a subject, by administering to the subject an IL-4 and IL-9 chimericmutein polypeptide antagonist, or an expressible polynucleotide encodingthe chimeric polypeptide, in an amount sufficient to reduce or inhibitspecific binding of IL-4, IL-9 IL-13, or a combination thereof to thereceptor, thereby ameliorating the pathologic condition in the subject.As used herein, the term “ameliorate,” when used in reference to apathologic condition, means that signs or symptoms associated with thecondition are lessened. The signs or symptoms to be monitored will becharacteristic of a particular pathologic condition and will be wellknown to skilled clinician, as will the methods for monitoring the signsand conditions. For example, where the pathologic condition is asthma,the skilled clinician will known that amelioration can be identified bydetecting improvements in parameters such as airwayhyper-responsiveness, and/or in airway inflammation including, forexample, by detecting mast cell, eosinophil and/or lymphocyterecruitment. Amelioration of a pulmonary disorder such as asthma,bronchitis, and the like can be determined, for example, by the subjectindicating that breathing is easier following administration of thechimeric polypeptide or encoding polynucleotide.

As disclosed herein, the methods of the invention provide a means toreduce or inhibit the responsiveness of cells to cytokines, particularlyIL-4, IL-9 and/or IL-13. As used herein, the term “responsiveness ofcells to a cytokine” means that the cells referred to express IL-4, IL-9and/or IL-13 receptors, which specifically bind IL-4, IL-9 and/or IL-13and mediate an effect on or by the cell. The effect can be any cellulareffect associated with cytokine binding including, for example, cellproliferation and protein expression. The terms “reduce or inhibit” areused together herein because it is recognized that, in some cases, thelevel of cell responsiveness, upon contact with a chimeric polypeptideantagonist of the invention, can be reduced below a level that can bedetected by a particular assay. As such, it may not be determinableusing such an assay as to whether expression of a protein by the cell isreduced below the level of detection of the assay, or is completelyinhibited. Regardless, however, a change will be measurable as comparedto the level of the responsiveness of the cell in the absence of thechimeric polypeptide antagonist.

A pathologic condition amenable to amelioration according to the presentmethods include, for example, pulmonary disorders such as asthma andchronic obstructive pulmonary disease (e.g., emphysema and chronicbronchitis), allergic inflammatory responses, and cancers (e.g.,leukemias and lymphomas). Features of asthma, for example, includerecurrent episodes of respiratory symptoms; variable airflow obstructionthat is often reversible, either spontaneously or with treatment;presence of airway hyper-reactivity; and chronic airway inflammation inwhich many cells and cellular elements, including, for example, mastcells, eosinophils, T lymphocytes, macrophages, neutrophils, andepithelial cells, are involved (see National Heart, Lung, and BloodInstitute: National Asthma Education and Prevention Program. ExpertPanel Report Guidelines for the diagnosis and management of asthma. J.Allergy Clin. Immunol. 88:425-534, 1991; National Heart, Lung, and BloodInstitute National Asthma Education Program Expert Panel Report II:Guidelines for the diagnosis and management of asthma; 1997. NIHPublication No. 97-4051A) While all of these features need not bepresent in any given asthmatic patient, and an absolute “minimumcriteria” to establish a diagnosis of asthma has not been or widelyagreed upon, the presence of airway hyper-reactivity is a common findingin patients with current symptoms and active asthma.

Asthma severity is graded into four categories based on the frequency ofsymptoms, peak flows, and the need for inhaled beta agonists: mildintermittent, mild persistent, moderate persistent, and severepersistent (Kavuru et al., “Asthma” (The Cleveland Clinic, January2003), available on the world wide web (www) at“clevelandclinicmeded.com/diseasemanagement/pulmonary/asthma/asthma.htm”).Hyperinflation, the most common finding on a chest radiograph, has nodiagnostic or therapeutic value. A chest radiograph should not beobtained unless complications of pneumonia, pneumothorax, or anendobronchial lesion are suspected. The correlation of severity betweenacute asthma and arterial blood gases is poor. Mild-to-moderate asthmais typically associated with respiratory alkalosis and mild hypoxemia onthe basis of ventilation-perfusion mismatching. Severe hypoxemia isquite uncommon in asthma. Normocapnia and hypercapnia do imply severeairflow obstruction, with FEV₁ usually less than 25% of the predictedvalue.

Airway hyperresponsiveness is a characteristic feature of asthma andconsists of an increased sensitivity of the airways to an inhaledconstrictor agonist, a steeper slope of the dose-response curve, and agreater maximal response to the agonist (Byrne and Inman, Chest, 2003).Measurements of airway responsiveness are useful in making a diagnosisof asthma, particularly in patients who have symptoms that areconsistent with asthma and who have no evidence of airflow obstruction.Certain inhaled stimuli, including, for example, environmentalallergens, increase airway inflammation and enhance airwayhyperresponsiveness. The changes in airway hyperresponsiveness inhealthy subjects are of much smaller magnitude than those seen whenasthmatic patients with persistent airway hyperresponsiveness. Thepathogenesis of asthma, and amelioration due to treatment according tothe present methods, can be followed by bronchoscopy, bronchoalveolarlavage, airway biopsy, measurement of airway gases, and other suchmethods known to the skilled clinician.

An amount of a chimeric polypeptide required to achieve a therapeuticbenefit can be determined using methods as disclosed herein or otherwiseknown in the art. A therapeutically effective dose refers to the amountof antagonist that is used to effectively treat a disorder as comparedwith the efficacy that is evident in the absence of the therapeuticallyeffective dose. A therapeutically effective dose can be estimatedinitially using an animal model (e.g., a rat, mouse, rabbit, dog, pig,or non-human primate model). An animal model also can be useful fordetermining an appropriate concentration range and route ofadministration, such that useful doses and routes for administrationthen can be identified for humans, for example, in clinical trials.

Therapeutic efficacy and toxicity, including, for example, an ED₅₀(i.e., a dose that is therapeutically effective in 50% of a specifiedpopulation) and LD₅₀ (the dose lethal to 50% of a population), of anIL-4 and IL-9 chimeric mutein antagonist can be determined usingstandard pharmaceutical procedures in cell cultures or experimentalanimals. The dose ratio of toxic to therapeutic effects (i.e., thetherapeutic index) it can be expressed as the ratio, LD₅₀/ED₅₀.Generally, pharmaceutical compositions that exhibit large therapeuticindices are preferred. The data obtained from animal studies is used informulating a range of dosages for human use. The dosage contained insuch compositions is preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage varies within this range depending upon the dosage form employed,sensitivity of the patient, and the route of administration.

An exact dosage for treating a human subject, for example, will bedetermined by the practitioner, in light of factors related to thepatient who requires treatment. Dosage and administration are adjustedto provide sufficient levels of the antagonist or to maintain thedesired effect. Factors that can be taken into account include theseverity of the disease state, general health of the subject, age,weight, and gender of the subject, diet, time and frequency ofadministration, drug combination(s), reaction sensitivities, andtolerance/response to therapy. Long-acting pharmaceutical compositionscan be administered every 3 to 4 days, every week, or once every twoweeks depending on the half-life and clearance rate of the particularformulation. Effective in vivo dosages of an antagonist of the inventioncan be in the range of about 0.1 to 50 mg/kg, including, for example,about 1 μg/kg to about 5 mg/kg, or about 10 μg/kg to about 1 mg/kg, orabout 100 to 250 μg/kg of patient body weight.

The following examples are intended to illustrate but not limit theinvention.

Example 1 IL-4 and IL-9 Chimeric Mutein Antagonists Inhibit CytokineInduced Cell Proliferation

This example demonstrates that an IL-4 and IL-9 chimeric muteinantagonists can inhibit cell proliferation due to IL-4 and/or IL-9.

Sequences

Exemplary polynucleotides encoding polyhistidine tagged IL-4RA/IL-9RA(SEQ ID NO:1) and IL-9RA/IL-4RA (SEQ ID NO:3) chimeric muteinantagonists, containing a TEV protease cleavage site between the tag andthe IL-4 and IL-9 components, are provided, as are the encodedpolypeptides (SEQ ID NOS:2 and 4, respectively). Also provided are anIL-9RA/IL-4RA chimeric polypeptide lacking a signal sequence (SEQ IDNO:5); and a polynucleotide sequence encoding an IL-9RA/IL-4RA chimericmutein antagonist, including a signal sequence (SEQ ID NO:6), and theencoded polypeptide, including the signal sequence (SEQ ID NO:7).

Protein Purification

Purification of the polypeptides is exemplified by IL-4RA purification.Anti-IL4 antibody column was stored in 20% ethanol at 4° C. The anti-IL4antibody column was used, and was equilibrated in running buffer at roomtemperature. A flow rate of about 2 ml per minute for a 1.5 cm diametercolumn was used. Buffers were as follows: “Running buffer” was phosphatebuffered saline (PBS); “Elution buffer” was 200 mM Glycine, pH2.8;“Neutralization buffer” was a saturated solution of Tris base; “Cleaningsolution: was 24% ethanol, 2% acetic acid.

Purification of the IL-4RA polypeptide was performed by running 3 columnvolumes of cleaning solution through the column, followed by 10 columnvolumes of PBS. The filtered sample was loaded, then 10 column volumesof PBS were passed over the column. IL-4RA was eluted using the elutionbuffer; the solution was neutralized immediately after collection with afew drops of Tris base. These steps were repeated for each purificationrun.

Purification of a HisTev tagged IL-9RA and IL-4RA chimeric receptorantagonist was performed using a chelating resin (e.g., PharmaciaChelating SEPHAROSE resin) charged with a metal ion (e.g., nickel). Thechelating resin was equilibrated in PBS (pH 7.0) supplemented to 500 mMNaCl to prevent non-specific binding of the tagged protein. Supernatantof cell cultures expressing the chimeric receptor antagonist wereconcentrated using a spin column with a molecular weight cut-offof >30,000 Daltons, then diluted 20-fold into PBS (pH 7.2) andconcentrated a second time. A second dilution was performed and thesupernatant was concentrated a third time to achieve about a 400-foldbuffer exchange. The concentrated and exchanged supernatant was appliedto an immobilized metal ion chromatography (IMAC) column, and washedwith several column volumes of equilibration buffer. Non-tagged proteinimpurities that bound to the column were washed with equilibrationbuffer adjusted to 10-20 mM imidazole (pH 7.5) HisTev tagged IL-9RA andIL-4RA chimeric receptor antagonist then was eluted from the columnsuing PBS adjusted to 250 mM imidazole (pH 7.5).

TF-1 Cell Proliferation Assay for IL-4 and IL-13 Antagonism

TF-1 cells are a erythroleukemia cell line that respond to severalpro-inflammatory cytokines by proliferation and, therefore, can be usedto assess cytokine bioactivity. The proliferative response of TF-1 cellsto IL-4 (0.5 ng/ml, 0.033 nM) or IL-13 (5 ng/ml, 0.416 nM) was used toassess the functional antagonistic activity of His6TEV-IL-4RA/IL-9RA(SEQ ID NO:2) and His6TEV-IL-9RA/IL-4RA (SEQ ID NO:4) chimericpolypeptides. It should be noted that the chimeric antagonistpolypeptides were expressed in cells, which post-translationally removethe signal peptide. As such, while reference is made in this Example toHis6TEV-IL-4RA/IL-9RA (SEQ ID NO:2) and His6TEV-IL-9RA/IL-4RA (SEQ IDNO:4), the chimeric polypeptides used in the experiments lack the signalpeptide, which is shown as amino acid residues 1 to 18 in each of SEQ IDNOS:2 and 4.

TF-1 cells were cultured for 3 days in 96 well plates (1×10⁴/well, 100μl volume) in RPMI +10% serum with or without IL-4 or IL-13 andHis6TEV-IL-4RA/IL-9RA (SEQ ID NO:2) or His6TEV-IL-9RA/IL-4RA (SEQ IDNO:4). GM-CSF treatment was used as a positive control. Twenty-fourhours before the final reading, 10 μl AlamarBlue™ dye (10% volume) wasadded to each well. Fluorescence was determined at 530/590 nm using aWALLAC Victor 2 spectrofluorimeter. The “Inhibitory Concentration 50%”(IC₅₀) was calculated based on dose titration of the candidateantagonistic molecules. IC₅₀ values obtained using His6TEV-IL-4RA/IL-9RA(SEQ ID NO:2) and His6TEV-IL-9RA/IL-4RA (SEQ ID NO:4) in the presence ofIL-4 or IL-13 are shown in Table 1 (below).

In further experiments, the ability of an untagged IL-9RA/IL-4RA (see,e.g., SEQ ID NOS:5 and 7; mutein antagonists lacking and containing thesignal peptide, respectively) to inhibit IL-4 induced proliferation ofTF-1 cells. As shown in Table 1, the untagged IL-9RA/IL-4RA chimericpolypeptide had an IC₅₀ of 0.6237 nM (n=3).

These results demonstrate that IL-4RA and IL-9RA chimeric muteinpolypeptides act as antagonists of IL-4 and/or IL-13 induced cellproliferation of erythroleukemia cells.

Mo7e Cell Proliferation Assay for IL-4 and IL-9 Antagonism

Mo7e cells are a megakaryoblastic leukemia cell line, which, like theTF-1 cells, respond to a number of pro-inflammatory cytokines viaproliferation. Mo7e cells also exhibit a proliferative response to IL-9in addition to IL-4. The proliferative response of Mo7e cells to IL-9(0.25 ng/ml, 17.8 μM)and IL-4 (0.5 ng/ml, 0.033 nM) was also examined toassess the functional antagonistic activity of His6TEV-IL-4RA/IL-9RA(SEQ ID NO:2) and His6TEV-IL-9RA/IL-4RA (SEQ ID NO:4) chimericpolypeptides. The Mo7e cell line was obtained from the Deutsche Sammlungvon Mikroorganismen and Zellkulturen GmbH (Braunschweig, Germany) andmaintained in RPMI-1640 with 10% fetal calf serum (RPMI-10), 10 ng/mlGM-CSF and penicillin.

Prior to experiments, Mo7e cells were starved of GM-CSF overnight andplated at 10⁴ cells/well in RPMI-10 cell culture media at a total volumeof 100 ul/well. Cells were treated with His6TEV-IL-4RA/IL-9RA (SEQ IDNO:2) or His6TEV-IL-9RA/IL-4RA (SEQ ID NO:4) plus wild-type human IL-9or IL-4 for 3 days. GM-CSF treatment was used as a positive control.Twenty-four hours before the final reading, 10 μl AlamarBlue™ dye (10%vol) was added to each well. Fluorescence was determined at 530/590 nmusing a WALLAC Victor 2 spectrofluorimeter. The IC₅₀ was calculatedbased on dose titration of the candidate antagonistic molecules. TheIC₅₀ values obtained using His6TEV-IL-4RA/IL-9RA (SEQ ID NO:2) andHis6TEV-IL-9RA/IL-4RA (SEQ ID NO:4) chimeric polypeptides in thepresence of wild-type IL-9 or wild-type IL4 are shown in Table 2(below).

The ability of untagged IL-9RA/IL-4RA (see, e.g., SEQ ID NOS:5 and 7) toinhibit IL-9 induced proliferation of Mo7e cells also was examined. Asshown in Table 2, the untagged IL-9RA/IL-4RA chimeric polypeptide had anIC₅₀ of 29.625 nM (n=2).

These results demonstrate that IL-4RA and IL-9RA chimeric muteinpolypeptides act as antagonist of IL-4 and/or IL-9 induced cellproliferation of megakaryoblastic leukemia cells.

Primary T-Cell Proliferation Assay

T-cells are a key component of an allergic immune response,orchestrating the response of several inflammatory cell pathways, andare involved in the pathogenesis of asthma in humans. The proliferativeresponse of primary T-cells to IL-4 was evaluated following pretreatmentof the cells with His6TEV-IL-4RA/IL-9RA (SEQ ID NO:2) orHis6TEV-IL-9RA/IL-4RA (SEQ ID NO:4) chimeric polypeptide. T-cells wereisolated from peripheral blood and treated with PHA-P for 4 days toinduce blast formation. The cells were washed and seeded in 96-wellplates (10⁵ cells per well). Phytohemmagluttinin (PHA) T-cell blastswere stimulated for 3 days with IL-4 (10 ng/ml, 0.667 nM) in thepresence of varying concentrations of the chimeric mutein antagonistpolypeptides. The incorporation of tritiated thymidine in the last 20hours of incubation was used as an indicator of proliferation.

The IC₅₀ values obtained using His6TEV-IL-4RA/IL-9RA (SEQ ID NO:2) andHis6TEV-IL-9RA/IL-4RA (SEQ ID NO:4) in the presence of wild-type IL-4are shown in Table 3 (below). These results demonstrate that IL-4RA andIL-9RA chimeric mutein polypeptides acts as antagonist of IL-4 inducedT-cell proliferation.

Primary B-Cell Proliferation Assay

T-cells affect B-cell proliferation and differentiation. B-cells areresponsible for the production of antibody (as differentiated plasmacells) and are inextricably linked to T-cell activity in the asthmaticpatient. The proliferative response of primary B-cells to IL-4 wasexamined following His6TEV-IL-4RA/IL-9RA (SEQ ID NO:2) andHis6TEV-IL-9RA/IL-4RA (SEQ ID NO:4). B-cells were isolated fromperipheral blood and treated with anti-CD40 MAb. The cells were seededin 96-well plates (10⁵ cells per well) and stimulated for 3 days withIL-4 (10 ng/ml, 0.667 nM) in the presence of varying concentrations ofmutein antagonist molecules. The incorporation of tritiated thymidine inthe last 20 hours of incubation was used as an indicator ofproliferation.

The IC₅₀ values obtained using His6TEV-IL-4RA/IL-9RA (SEQ ID NO:2) andHis6TEV-IL-9RA/IL-4RA (SEQ ID NO:4) in the presence of wild-type IL-4are shown in Table 3 (below). These results demonstrate that IL-4RA andIL-9RA chimeric mutein polypeptides acts as antagonist of IL-4 inducedB-cell proliferation.

TABLE 1 IL-9 and IL-4 Chimeric Antagonist Mutein Bioactivity Evaluationin TF-1 Cell Proliferation Assay TF-1/IL4 TF-1/IL13 IC_(50,) nM IC_(50,)nM His6TEV-IL-  8.01 ± 1.73 (n = 3) 20.14 ± 0 (n = 1) 9RA/IL-4RA (4*)His6TEV-IL- 20.66 ± 1.89 (n = 3)  56.2 ± 0 (n = 1) 4RA/IL-9RA (3)IL-9RA/IL-4RA (5) 0.6237 nM (n = 3) *number in parentheses is SEQ ID NO:

TABLE 2 IL-9 and IL-4 Chimeric Antagonist Mutein Bioactivity Evaluationin Mo7e Cell Proliferation Assay Mo7e/IL9 Mo7e/IL4 IC_(50,) nM IC_(50,)nM His6TEV-IL-  83.62 ± 24.73 (n = 2) 10.13 ± 0.18 (n = 2) 9RA/IL-4RA(4*) His6TEV-IL- 570.75 ± 163.7 (n = 2) 15.24 ± 1.36 (n = 2) 4RA/IL-9RA(3) IL-9RA/IL-4RA (5) 11.49 nM (n = 1) *number in parentheses is SEQ IDNO:

TABLE 3 IL-9 and IL-4 Chimeric Antagonist Mutein Bioactivity EvaluationPrimary T-Cell and B-Cell Proliferation Assay B-cell T-cell blastIC_(50,) nM IC_(50,) nM His6TEV-IL- 10.62 ± 5.71 (n = 3)  33.98 ± 7.02(n = 4) 9RA/IL-4RA (4*) His6TEV-IL- 16.34 ± 10.55 (n = 3) 103.89 ± 46.99(n = 4) 4RA/IL-9RA (3) *number in parentheses is SEQ ID NO:

REFERENCES CITED

Each of the following articles is incorporated herein by reference.

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Henderson et al., J. Immunol. 164: 1086-1095, 2000.

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Although the invention has been described with reference to the aboveexample, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

1. A chimeric polypeptide, comprising an interleukin-4 (IL-4) muteinreceptor antagonist operatively linked to an interleukin-9 (IL-9) muteinreceptor antagonist, wherein the chimeric polypeptide can reduce orinhibit the association of an interleukin with an IL-4 receptor, an IL-9receptor, an interleukin-13 (IL-13) receptor, or a combination thereof.2. A method of reducing or inhibiting cytokine responsiveness of a cell,comprising contacting the cell with a chimeric polypeptide of claim 1.3. The method of claim 2, wherein the cytokine responsiveness of thecell comprises proliferation of the cell, protein expression by thecell, or a combination thereof.
 4. The method of claim 2, wherein thecytokine responsiveness comprises IL-4 responsiveness, IL-9responsiveness, IL-13 responsiveness, or a combination thereof.
 5. Themethod of claim 2, wherein cell comprises a lymphocyte, apolymorphonuclear leukocyte, a monocyte, or a combination thereof. 6.The method of claim 5, wherein the cell comprises a B lymphocyte, a Tlymphocyte, an eosinophil, or a combination thereof.
 7. The method ofclaim 2, wherein the cell comprises a leukemia cell.
 8. The method ofclaim 7, wherein the leukemia cell comprises an erythroleukemia cell ora megakaryoblastic leukemia cell.
 9. A method of ameliorating apathologic condition associated with cells expressing at least onereceptor selected from an IL-4 receptor, an IL-9 receptor, or an IL-13receptor in a subject, comprising administering to the subject achimeric polypeptide of claim 1 in an amount sufficient to reduce orinhibit specific binding of IL-4, IL-9 IL-13, or a combination thereofto the receptor, thereby ameliorating the pathologic condition in thesubject.
 10. The method of claim 9, wherein the subject is a mammal. 11.The method of claim 9, wherein the subject is a human.
 12. The method ofclaim 9, wherein the pathologic condition comprises asthma, chronicobstructive pulmonary disease, an allergic inflammatory response, or acombination thereof.
 13. The method of claim 12, wherein the chronicobstructive pulmonary disease comprises emphysema or chronic bronchitis.